WO2018207827A1 - ポリペプチド溶液、及びポリペプチド繊維の製造方法、並びに人造ポリペプチド - Google Patents

ポリペプチド溶液、及びポリペプチド繊維の製造方法、並びに人造ポリペプチド Download PDF

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WO2018207827A1
WO2018207827A1 PCT/JP2018/017953 JP2018017953W WO2018207827A1 WO 2018207827 A1 WO2018207827 A1 WO 2018207827A1 JP 2018017953 W JP2018017953 W JP 2018017953W WO 2018207827 A1 WO2018207827 A1 WO 2018207827A1
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Prior art keywords
polypeptide
keratin
amino acid
acid sequence
solution
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PCT/JP2018/017953
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English (en)
French (fr)
Japanese (ja)
Inventor
あゆみ 安部
森田　啓介
建始 倉知
佑之介 安部
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Ｓｐｉｂｅｒ株式会社
小島プレス工業株式会社
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Application filed by Ｓｐｉｂｅｒ株式会社, 小島プレス工業株式会社 filed Critical Ｓｐｉｂｅｒ株式会社
Priority to CN201880025883.8A priority Critical patent/CN110546264A/zh
Priority to EP18797659.2A priority patent/EP3626824A4/en
Priority to US16/611,315 priority patent/US20200165311A1/en
Priority to JP2019517665A priority patent/JP7270978B2/ja
Publication of WO2018207827A1 publication Critical patent/WO2018207827A1/ja

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4741—Keratin; Cytokeratin
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/06—Wet spinning methods
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2203/00—Applications
    • C08L2203/12—Applications used for fibers

Definitions

  • the present invention relates to a polypeptide solution, a method for producing a polypeptide fiber, and an artificial polypeptide.
  • Patent Document 1 and Patent Document 2 An attempt has been made to dissolve keratin, which is a major component of hair, and regenerate and reuse the fiber (Patent Document 1 and Patent Document 2). More specifically, pulverized keratin is used as a feed or fertilizer, a cosmetic base material, a surfactant suitable for an ecosystem, a medical polymer material, and the like (Patent Document 3). On the other hand, since natural animal hair has a complex structure (Non-patent Document 1), no attempt has been made to produce fibers similar to artificial hair.
  • Keratin can be classified into type I (acidic) and type II (neutral or basic) keratin by isoelectric point (Non-patent Document 1).
  • Type I keratin can be further classified into human type I epithelial keratin (K9-K28), human type I hair keratin (K31-K40), non-human type I epithelium and hair keratin (K41-K70) (non- Patent Document 2).
  • K26 Keratin 26
  • K25 and K27 classified as cluster Ia have high bootstrap values and are known to be closely related in amino acid sequence.
  • K26 is known to have high sequence similarity across species (Non-patent Document 2).
  • JP 2010-236149 A Japanese Unexamined Patent Publication No. 2016-160211 JP 05-170926 A
  • Non-Patent Document 2 the amino acid sequence has been clarified by gene analysis of keratin, but even this keratin has not been artificially produced.
  • a polypeptide comprising a polypeptide containing an amino acid sequence derived from type I keratin belonging to cluster Ia, dissolved in a solvent containing formic acid or a solvent containing an aprotic polar solvent having a dipole moment of 3.0D or more and an inorganic salt solution.
  • the thiols are at least selected from the group consisting of dithiothreitol, ⁇ -mercaptoethanol, 3-mercapto-1,2-propanediol, 1,2-ethanethiol, thioglycolic acid and ammonium thioglycolate (ATG).
  • the polypeptide solution according to [3] which is one type.
  • the aprotic polar solvent having a dipole moment of 3.0D or more is dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, 1,3-dimethyl-2-imidazolidone, N-methyl-2-
  • the inorganic salt is at least one selected from the group consisting of alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates and thiocyanates, according to any one of [1] to [5].
  • Polypeptide solution is at least one selected from the group consisting of alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates and thiocyanates, according to any one of [1] to [5].
  • An expression vector comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence derived from type I keratin belonging to cluster Ia, and one or more regulatory sequences operably linked to the nucleic acid sequence.
  • An artificial polypeptide comprising an amino acid sequence derived from type I keratin belonging to cluster Ia.
  • polypeptide solution suitable for producing a polypeptide fiber containing a keratin-derived polypeptide.
  • method for producing a polypeptide fiber using the polypeptide solution it is possible to provide a polypeptide solution suitable for producing a polypeptide fiber containing a keratin-derived polypeptide.
  • the polypeptide solution according to this embodiment is obtained by dissolving a polypeptide containing an amino acid sequence derived from type I keratin belonging to cluster Ia in a solvent.
  • the solvent may be a solvent containing formic acid or a solvent containing an aprotic polar solvent having a dipole moment of 3.0 D or more and an inorganic salt.
  • the “artificial polypeptide” is a term for distinguishing from a naturally-occurring polypeptide, and includes an artificially produced polypeptide (for example, by a genetic recombination method). In this sense, the artificial polypeptide may be a polypeptide having the same amino acid sequence as the natural amino acid sequence or a polypeptide having an amino acid sequence different from the natural amino acid sequence.
  • the polypeptide according to the present invention includes an amino acid sequence derived from type I keratin belonging to cluster Ia (hereinafter also simply referred to as “polypeptide of the present invention”).
  • the cluster Ia is obtained by analyzing the amino acid sequence of keratin by the neighbor-joining method (Neighbor-joining method) and performing goat (Capra hircus) keratin 25 (K25) and keratin 26 ( K26) and type I keratin clusters to which sheep (Ovis aries) keratin 25 (K25) and keratin 27 (K27) belong.
  • amino acid sequence derived from type I keratin belonging to cluster Ia means an amino acid sequence of a natural type I keratin classified into cluster Ia when cluster analysis is performed by the neighbor binding method, and the natural type It includes an amino acid sequence obtained by modifying the amino acid sequence of I keratin.
  • “including an amino acid sequence” includes a case where the amino acid sequence is comprised and a case where another amino acid sequence is further added to the amino acid sequence.
  • (natural) type I keratin belonging to cluster Ia include keratin 25 (K25), keratin 26 (K26) and keratin 27 (K27), with K26 being preferred.
  • FIG. 1 is an alignment diagram comparing the amino acid sequences of K26 derived from various organisms.
  • goats Capra hircus, NP_00127262643.1
  • cattle Bos taurus, NM_001099096.1
  • humans Homo sapiens, BC132951.1
  • mice Mus musculus, BC116672.1
  • K26 derived from rat has extremely high sequence identity (in parentheses is the number of the GenBank database) and is considered to have very similar properties as a protein.
  • the shaded residues indicate residues that match the goat K26 sequence.
  • An asterisk below the alignment indicates sequence identity.
  • a colon indicates a conservative substitution, and a dot indicates a non-conservative substitution.
  • the polypeptide of the present invention has substitution, deletion, insertion and / or addition of one or a plurality of amino acid residues in the amino acid sequence of (natural) type I keratin belonging to cluster Ia as long as it possesses keratin properties. It may be one containing an amino acid sequence that has been modified correspondingly (hereinafter also referred to as “modified polypeptide”).
  • modified polypeptide examples include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and 85% or more, preferably 90% or more of these amino acid sequences,
  • a modified polypeptide comprising an amino acid sequence having a sequence identity of 95% or more, more preferably 98% or more, and even more preferably 99% or more can be mentioned.
  • the modified polypeptide according to this embodiment may include a tag sequence at one or both of the N-terminus and the C-terminus. By including the tag sequence, the polypeptide can be isolated, immobilized, detected and visualized.
  • tag sequences include affinity tags that use specific affinity (binding property, affinity) with other molecules.
  • affinity tag include a histidine tag (His tag).
  • His tag is a short peptide with about 4 to 10 histidine residues, and has the property of specifically binding to metal ions such as nickel. Therefore, isolation of the polypeptide by metal chelating chromatography. Can be used.
  • Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 17 (amino acid sequence containing a His tag).
  • GST glutathione-S-transferase
  • MBP maltose binding protein
  • an “epitope tag” using an antigen-antibody reaction can also be used as a tag sequence.
  • a peptide (epitope) exhibiting antigenicity as a tag sequence
  • an antibody against the epitope can be bound.
  • the epitope tag include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, and FLAG tag.
  • a tag sequence that can be separated by a specific protease can also be used.
  • the polypeptide from which the tag sequence has been cleaved can also be recovered.
  • Specific examples of the modified polypeptide containing such a tag sequence include the amino acid sequences represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, Modified amino acids having amino acid sequences having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and even more preferably 99% or more of these amino acid sequences.
  • Peptides can be mentioned.
  • a modified polypeptide fused with a spider protein having high toughness and a poly A sequence that is one of the features of the spider protein are inserted.
  • Modified polypeptides having the amino acid sequence described above can also be used.
  • the modified polypeptide specifically includes the amino acid sequences represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, and 85% or more, preferably 90% or more, and more than these amino acid sequences. May include a modified polypeptide comprising an amino acid sequence having a sequence identity of 95% or more, more preferably 98% or more, and even more preferably 99% or more.
  • the artificial polypeptide according to this embodiment is transformed with, for example, an expression vector having a nucleic acid sequence encoding the polypeptide of the present invention and one or more regulatory sequences operably linked to the nucleic acid sequence. It can be produced by culturing a host, growing it, and inducing expression of the polypeptide.
  • nucleic acid The method for producing a nucleic acid encoding the polypeptide of the present invention is not particularly limited. For example, using a gene encoding natural keratin, amplification and cloning by polymerase chain reaction (PCR) or the like, and if necessary, modification by genetic engineering techniques, or chemical synthesis, Nucleic acids can be produced.
  • PCR polymerase chain reaction
  • the chemical synthesis method of the nucleic acid is not particularly limited, and for example, based on the amino acid sequence information of keratin obtained from the NCBI web database or the like, AKTA oligopilot plus 10/100 (manufactured by GE Healthcare Japan, Inc.)
  • the gene encoding the polypeptide of the present invention can be chemically synthesized by a method of linking oligonucleotides automatically synthesized with a device such as) by a known genetic engineering technique such as PCR.
  • the regulatory sequence is a sequence that controls the expression of the polypeptide in the host (eg, promoter, enhancer, ribosome binding sequence, transcription termination sequence, etc.), and can be appropriately selected depending on the type of the host.
  • an inducible promoter that functions in a host cell and can induce expression of a polypeptide may be used.
  • An inducible promoter is a promoter that can control transcription by the presence of an inducer (expression inducer), the absence of a repressor molecule, or physical factors such as an increase or decrease in temperature, osmotic pressure or pH value. .
  • the type of expression vector can be appropriately selected according to the type of host, such as a plasmid vector, a viral vector, a cosmid vector, a fosmid vector, an artificial chromosome vector, and the like.
  • a vector that can replicate autonomously in a host cell or can be integrated into a host chromosome and contains a promoter at a position where a nucleic acid encoding a polypeptide can be transcribed is preferably used.
  • any of prokaryotes and eukaryotes such as yeast, filamentous fungi, insect cells, animal cells and plant cells can be preferably used.
  • prokaryotic hosts include bacteria belonging to the genus Escherichia, Brevibacillus, Serratia, Bacillus, Microbacterium, Brevibacterium, Corynebacterium, Pseudomonas and the like.
  • microorganisms belonging to the genus Escherichia include Escherichia coli.
  • microorganisms belonging to the genus Brevibacillus include Brevibacillus agri.
  • microorganisms belonging to the genus Serratia include Serratia liqufaciens and the like.
  • microorganisms belonging to the genus Bacillus include Bacillus subtilis.
  • microorganisms belonging to the genus Microbacterium include microbacterium / ammonia film.
  • microorganisms belonging to the genus Brevibacterium include Brevibacterium divaricatam.
  • microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes.
  • microorganisms belonging to the genus Pseudomonas include Pseudomonas putida.
  • examples of an expression vector for introducing a nucleic acid encoding the polypeptide of the present invention include pBTrp2 (manufactured by Boehringer Mannheim Co.), pGEX (manufactured by Pharmacia Co., Ltd.), pUC18, pBluescriptII, pSupex, Examples include pET22b, pCold, pUB110, and pNCO2 (Japanese Patent Laid-Open No. 2002-238696).
  • Examples of eukaryotic hosts include yeast and filamentous fungi (molds, etc.).
  • yeast include yeasts belonging to the genus Saccharomyces, Pichia, Schizosaccharomyces and the like.
  • Examples of the filamentous fungi include filamentous fungi belonging to the genus Aspergillus, the genus Penicillium, the genus Trichoderma and the like.
  • examples of an expression vector for introducing a nucleic acid encoding the polypeptide of the present invention include YEP13 (ATCC37115), YEp24 (ATCC37051) and the like.
  • a method for introducing the expression vector into the host cell any method can be used as long as it is a method for introducing DNA into the host cell.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]
  • electroporation method electroporation method
  • spheroplast method protoplast method
  • lithium acetate method lithium acetate method
  • competent method and the like.
  • the artificial polypeptide according to the present embodiment is obtained by, for example, culturing a host transformed with an expression vector in a culture medium, producing and accumulating the artificial polypeptide in the culture medium, and isolating the artificial polypeptide from the culture medium. It can be collected by purification.
  • the method for culturing a host in a culture medium can be performed according to a method usually used for culturing a host.
  • the culture medium contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host. Any natural or synthetic medium may be used as long as it is an efficient medium.
  • Any carbon source may be used as long as the transformed host can be assimilated, such as glucose, fructose, sucrose, and carbohydrates such as molasses, starch and starch hydrolysate, acetic acid and propionic acid.
  • Organic acids such as ethanol and alcohols such as ethanol and propanol can be used.
  • the nitrogen source include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
  • inorganic salts for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.
  • Cultivation of prokaryotes such as E. coli or eukaryotes such as yeast can be performed under aerobic conditions such as shaking culture or deep aeration and agitation culture.
  • the culture temperature is, for example, 15 to 40 ° C.
  • the culture time is usually 16 hours to 7 days.
  • the pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0.
  • the pH of the culture medium can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • antibiotics such as ampicillin and tetracycline may be added to the culture medium as necessary.
  • an inducer may be added to the culture medium as necessary.
  • the host transformed with an expression vector using the trp promoter is cultured with isopropyl- ⁇ -D-thiogalactopyranoside or the like.
  • indoleacrylic acid or the like may be added to the culture medium.
  • Isolation and purification of artificial polypeptide can be performed by a commonly used method. For example, when the artificial polypeptide is secreted outside the host cell, the artificial polypeptide can be recovered from the culture supernatant.
  • a culture supernatant is obtained by treating the culture containing the host cells by a technique such as centrifugation, and a method commonly used for isolation and purification of known polypeptides from the culture supernatant, for example, , Solvent extraction method, salting out method using ammonium sulfate, desalting method, precipitation method using organic solvent, anion exchange using resin such as diethylaminoethyl (DEAE) -sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei Co., Ltd.) Chromatography method, cation exchange chromatography method using resin such as S-Sepharose FF (Pharmacia Co., Ltd.), hydrophobic chromatography method using resin such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve Method, affinity chromatography method, chromatofocusing method, isoelectric focusing The method such as electrophoresis and used alone or in combination, such as electrophores
  • the host cell when the artificial polypeptide is expressed in a dissolved state in the host cell, the host cell is recovered by centrifugation after culturing and suspended in an aqueous buffer, followed by an ultrasonic crusher, a French press. Then, the host cells are disrupted with a Manton Gaurin homogenizer, dynomill or the like to obtain a cell-free extract. A purified sample can be obtained from the supernatant obtained by further centrifuging the cell-free extract by using the same isolation and purification method as described above.
  • the host cell when the artificial polypeptide is expressed in the form of insoluble matter as granules in the host cell, the host cell is recovered and crushed and centrifuged as described above to obtain the precipitate fraction. Collect artificial polypeptide granules.
  • the recovered granules of the artificial polypeptide can be solubilized with a protein denaturant (hereinafter also simply referred to as “denaturant”).
  • the insoluble material is washed with a buffer solution such as 20 mM Tris-HCl buffer (pH 8.0), a base such as sodium hydroxide, an aqueous solution such as an alkali metal salt or alkaline earth metal salt, or an oxo acid. Also good.
  • a buffer solution such as 20 mM Tris-HCl buffer (pH 8.0), a base such as sodium hydroxide, an aqueous solution such as an alkali metal salt or alkaline earth metal salt, or an oxo acid. Also good.
  • the modifying agent examples include urea, thiourea, guanidinium chloride (guanidine hydrochloride), a surfactant and the like.
  • urea or thiourea the concentration is preferably 6 to 9M.
  • guanidinium chloride the concentration is preferably 4 to 8M.
  • the surfactant include SDS, and 1 to 2 g may be added to 1 g of protein.
  • a purified preparation can be obtained by the same isolation and purification method as described above.
  • a method in which the granules are dissolved in urea, and then the artificial polypeptide is precipitated and recovered with an organic solvent and purified for example, trichloroacetic acid (TCA) solution, guanidine hydrochloride solution, perchloric acid solution
  • TCA trichloroacetic acid
  • guanidine hydrochloride solution guanidine hydrochloride solution
  • perchloric acid solution an aqueous solution of a protein denaturant such as, an aqueous solution of one or more selected from acidic substances such as hydrochloric acid, sulfuric acid, acetic acid, and phosphoric acid can be used, and organic solvents such as acetone and ethanol can be used. Further, an organic solvent containing normal propyl alcohol, isopropyl alcohol or the like in the ethanol may be used.
  • the organic solvent can be added in an amount of 1 to 6 times, preferably 2 to 5 times, to the solution using urea.
  • the artificial polypeptide is purified and extracted from the wet or dry cells of Escherichia coli for expressing the artificial polypeptide according to the present embodiment using an aprotic polar solvent having a dipole moment of 3.0D or more.
  • the artificial polypeptide can be extracted from a wet cell or a dry cell of Escherichia coli that expresses the artificial polypeptide according to the present embodiment using a solvent containing formic acid.
  • Table 1 lists aprotic polar solvents having a dipole moment of 3.0D or more based on the solvent handbook (Kodansha Scientific, 2007).
  • aprotic polar solvents having a dipole moment of 3.0D or more dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMA), Examples include 1,3-dimethyl-2-imidazolidone (DMI), N-methyl-2-pyrrolidone (NMP), and acetonitrile.
  • DMSO dimethyl sulfoxide
  • DMF N-dimethylformamide
  • DMA N-dimethylacetamide
  • Examples include 1,3-dimethyl-2-imidazolidone (DMI), N-methyl-2-pyrrolidone (NMP), and acetonitrile.
  • the artificial polypeptide according to the present embodiment When the artificial polypeptide according to the present embodiment is expressed in the form of insoluble matter as a granule in a host cell, the insoluble matter is usually poorly soluble in the aprotic polar solvent. Therefore, by treating with the aprotic polar solvent, unnecessary components derived from host cells can be removed, and the purity of the granules can be improved.
  • the purity of the granule can be further improved by treating with the aprotic polar solvent.
  • the aprotic polar solvent may be added to the granules obtained from the host cells.
  • unnecessary components derived from host cells can be dissolved more efficiently by adding an inorganic salt to the aprotic polar solvent to such an extent that the granules do not dissolve.
  • inorganic salts include alkali metal halides, alkaline earth metal halides, alkaline earth metal nitrates, thiocyanates, perchlorates and the like.
  • alkali metal halide examples include potassium bromide, sodium bromide, lithium bromide, potassium chloride, sodium chloride, lithium chloride, sodium fluoride, potassium fluoride, cesium fluoride, potassium iodide, sodium iodide, A lithium iodide etc. can be mentioned.
  • alkaline earth metal halide examples include calcium chloride, magnesium chloride, magnesium bromide, calcium bromide, magnesium iodide, calcium iodide and the like.
  • alkaline earth metal nitrates examples include calcium nitrate, magnesium nitrate, strontium nitrate, and barium nitrate.
  • thiocyanate examples include sodium thiocyanate, ammonium thiocyanate, (guanidinium thiocyanate), and the like.
  • perchlorates examples include ammonium perchlorate, potassium perchlorate, calcium perchlorate, silver perchlorate, sodium perchlorate, and magnesium perchlorate.
  • the addition amount (wt) of the inorganic salt may be determined in accordance with the aprotic polar solvent to be used, and is not particularly limited.
  • the aprotic polar solvent for the total amount of the aprotic polar solvent (vol)
  • 0 to 3% (wt / vol) is preferable.
  • the treatment temperature for purifying the granules may be determined according to the concentration of the inorganic salt to be added, the type of the target artificial polypeptide, etc., and is not particularly limited, but is, for example, 10 to 60 ° C.
  • the temperature is preferably 10 to 50 ° C.
  • the treatment time is not particularly limited as long as the target artificial polypeptide does not dissolve, but it is 10 to 100 minutes in consideration of industrial production. It may be 10 to 60 minutes, more preferably 10 to 30 minutes.
  • the insoluble artificial polypeptide in the form of granules can be dissolved by using an aprotic polar solvent having a dipole moment of 3.0 D or more to which a solvent or inorganic salt containing formic acid is added at a higher concentration. And can be extracted from the host cell. Further, when the artificial polypeptide contains a cysteine residue, it is preferable to add a reducing agent to the solvent.
  • a solvent used in the purification of the granule can be mentioned as a suitable solvent.
  • inorganic salts used in the purification of the granules can be mentioned as suitable inorganic salts.
  • the inorganic salts mentioned above may be used alone or in combination of two or more.
  • the addition amount (wt) of the inorganic salt may be determined in accordance with the aprotic polar solvent to be used and is not particularly limited.
  • the total amount (vol) of the aprotic polar solvent thus, it is preferably 0.1 to 20% (wt / vol).
  • Examples of the reducing agent include dithiothreitol (DTT), ⁇ -mercaptoethanol (BME), 3-mercapto-1,2-propanediol, 1,2-ethanethiol, thioglycolic acid (TGA), thioglycolic acid
  • Examples include thiols such as ammonium (ATG), phosphine derivatives such as tris (2-carboxyethyl) phosphine hydrochloride (TCEP), tris (hydroxypropyl) phosphine (THPP), and sodium pyrosulfite (SM). .
  • the addition amount (wt) of the reducing agent is not particularly limited. For example, it is 0.1 to 10% (wt / vol) with respect to the total amount of the aprotic polar solvent (vol) used. Preferably, it is 1 to 5%.
  • the ratio of the artificial polypeptide (dry weight of host cell wt) to the added amount (vol) of the formic acid or the like or the aprotic polar solvent may be 1 to 20% (wt / vol), and 4 to 16%. Preferably, it is 6 to 12%.
  • the artificial polypeptide can be efficiently dissolved and extracted by heating.
  • the temperature for heating and dissolving may be determined according to the solvent to be added, but is usually 10 to 90 ° C, preferably 30 to 85 ° C, more preferably 40 to 80 ° C. .
  • the treatment time is not particularly limited as long as the artificial polypeptide can be sufficiently dissolved. However, considering industrial productivity and yield, it is usually 20 to 80 minutes, and 20 to 70 minutes. It is preferably 20 to 60 minutes.
  • Centrifugation conditions are not particularly limited, and examples include conditions at room temperature (20 ⁇ 5 ° C.) and 8000 ⁇ g to 15000 ⁇ g for 5 to 20 minutes.
  • filter filtration a solvent in which the polypeptide according to this embodiment is dissolved can be more efficiently recovered by using a filter aid such as celite and diatomaceous earth and a precoat agent in combination.
  • the insoluble matter may be separated twice or more.
  • the polypeptide solution according to this embodiment can be prepared as follows.
  • the preparation of the present invention is performed by adding the polypeptide of the present invention (for example, the artificial polypeptide according to the present embodiment) to a solvent obtained by adding an inorganic salt to an aprotic polar solvent having a dipole moment of 3.0D or more.
  • it can be prepared by adding the polypeptide of the present invention to a solvent containing formic acid.
  • a reducing agent to the solvent (in particular, a solvent obtained by adding an inorganic salt to an aprotic polar solvent).
  • this dissolved solution is prepared so as to be the following solution. It can be used as such a polypeptide solution.
  • the solvents described in the isolation and purification of artificial polypeptides can be mentioned as suitable solvents.
  • inorganic salts described in the isolation and purification of artificial polypeptides can be mentioned as suitable inorganic salts. These inorganic salts may be used alone or in combination of two or more.
  • an optimum amount may be determined depending on the aprotic polar solvent to be used, for example, 0.1 to 20% with respect to the aprotic polar solvent (vol) ( wt / vol), preferably 1 to 10% (wt / vol), more preferably 1 to 5% (wt / vol).
  • the reducing agents described in the isolation and purification of artificial polypeptides can be mentioned as suitable reducing agents.
  • the addition amount (wt) of the reducing agent with respect to the total amount of the solvent is preferably 0.1 to 10% (wt / vol), more preferably 1 to 5% with respect to the aprotic polar solvent (vol).
  • the solvent containing formic acid may be composed only of formic acid, and may contain other solvents, the above-described inorganic salts and / or reducing agents in addition to formic acid.
  • the ratio of the dry weight (wt) of the polypeptide of the present invention to the addition amount (vol) of formic acid or the like or an aprotic polar solvent may be 5 to 35% (wt / vol), and 10 to 30%. Preferably, it is 12 to 28%.
  • the polypeptide of the present invention can be efficiently dissolved by heating.
  • the temperature for heating and dissolving may be determined according to the solvent to be added, but may be 10 to 120 ° C, preferably 30 to 120 ° C, more preferably 40 to 120 ° C. preferable.
  • the treatment time (warming time) for heating is not particularly limited as long as the polypeptide of the present invention can be sufficiently dissolved, but usually 3 to 80 in view of industrial productivity and yield. Minutes, preferably 5 to 70 minutes, more preferably 5 to 60 minutes.
  • the polypeptide solution according to this embodiment can be used as a dope solution.
  • the dope solution is useful for spinning, cast film solution, and the like.
  • the dope solution may contain inevitable components such as impurities that have not been removed by purification of the artificial polypeptide.
  • the viscosity of the dope solution in spinning may be appropriately set according to the spinning method. For example, it is 100 to 15,000 cP (centipoise) at 35 ° C., more preferably 1,000 to 15,000 cP, and still more preferably 1,000. Up to 10,000 cP.
  • the adjustment of the viscosity of the polypeptide solution according to the present embodiment can be performed, for example, by adjusting the concentration and / or temperature of the polypeptide of the present invention in the solution.
  • the viscosity of the polypeptide solution can be measured using, for example, a trade name “electromagnetic spinning viscometer” manufactured by Kyoto Electronics Industry Co., Ltd.
  • the spinning method is not particularly limited as long as it is a method capable of spinning the polypeptide of the present invention, and examples thereof include dry spinning, melt spinning, wet spinning, and dry wet spinning.
  • dry spinning melt spinning
  • wet spinning dry wet spinning
  • dry wet spinning can be exemplified.
  • the polypeptide solution (dope solution) according to the present embodiment is extruded from a spinneret (nozzle) into a coagulating liquid (coagulating liquid tank), and the present invention is used in the coagulating liquid.
  • An undrawn yarn in the form of a yarn can be obtained by solidifying the polypeptide.
  • the coagulation liquid may be any solution that can be desolvated, and examples thereof include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone. Water may be appropriately added to the coagulation liquid.
  • the temperature of the coagulation liquid is preferably 5 to 30 ° C.
  • the extrusion speed is preferably 0.2 to 6.0 ml / hour per hole.
  • a more preferable extrusion rate is 1.4 to 4.0 ml / hour per hole.
  • the length of the coagulation bath may be long enough to efficiently remove the solvent, and is, for example, 200 to 500 mm.
  • the take-up speed of the undrawn yarn may be, for example, 1 to 20 m / min, and preferably 1 to 3 m / min.
  • the residence time may be, for example, 0.01 to 3 minutes, and preferably 0.05 to 0.15 minutes.
  • stretching pre-stretching
  • the coagulation liquid may be kept at a low temperature and taken up in an undrawn yarn state.
  • the coagulating liquid tank may be provided in multiple stages, and the stretching may be performed at each stage or a specific stage as required.
  • the take-up speed of the undrawn yarn is preferably 1 to 3 m / min.
  • the undrawn yarn (or predrawn yarn) obtained by the above method can be made into a drawn yarn (polypeptide fiber) through a drawing step.
  • Stretching methods include wet heat stretching, dry heat stretching, and solidification bath stretching.
  • Dry heat stretching can be performed using an electric tubular furnace, a dry heat plate, or the like.
  • the temperature may be, for example, 140 ° C. to 270 ° C., and preferably 160 ° C. to 230 ° C.
  • an undrawn yarn or predrawn yarn
  • an undrawn yarn can be drawn, for example, 0.5 to 8 times, and preferably 1 to 4 times.
  • Wet heat stretching and dry heat stretching may be performed independently, or may be performed in multiple stages or in combination. That is, the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by dry heat stretching, or the first stage stretching is performed by wet heat stretching, the second stage stretching is performed by wet heat stretching, and the third stage stretching is performed by dry heat stretching.
  • wet heat stretching and dry heat stretching can be appropriately combined.
  • the final draw ratio in the drawing step is, for example, 5 to 20 times, preferably 6 to 11 times that of the undrawn yarn (or predrawn yarn).
  • the artificial polypeptide fiber (artificial keratin fiber) formed from the artificial polypeptide according to the present invention is a fiber (long fiber, short fiber, multifilament, monofilament, etc.) or yarn (spun yarn, twisted yarn, false twisted yarn, processed yarn).
  • a fiber long fiber, short fiber, multifilament, monofilament, etc.
  • yarn spun yarn, twisted yarn, false twisted yarn, processed yarn.
  • the artificial polypeptide according to the present invention can also be applied to films, foams, granules (spheres or non-spheres, etc.), nanofibrils, gels (hydrogels, etc.), resins, and equivalents thereof. It can be produced according to the methods described in Japanese Patent Application Publication No. 2009-505668, Japanese Patent No. 5678283, Japanese Patent No. 4638735, and the like.
  • nucleic acid encoding artificial polypeptide and construction of expression vector Nucleic acids encoding artificial polypeptides having the amino acid sequences shown in SEQ ID NOs: 6 to 16 were respectively synthesized. Each synthesized nucleic acid was cloned into a cloning vector (pUC118). Thereafter, each was recombined with the protein expression vector pET-22b (+) to obtain an expression vector.
  • An artificial polypeptide (PRT798) having the amino acid sequence represented by SEQ ID NO: 6 is represented by SEQ ID NO: 17 at the N-terminal of the amino acid sequence of Capra hircus (GenBank accession number: NP_00127262643.1) represented by SEQ ID NO: 1. It has an amino acid sequence to which an amino acid sequence (tag sequence and hinge sequence) is added.
  • the artificial polypeptide (PRT800) having the amino acid sequence represented by SEQ ID NO: 7 has an amino acid sequence in which the cysteine of PRT798 (SEQ ID NO: 6) is substituted with serine.
  • the artificial polypeptide (PRT801) having the amino acid sequence represented by SEQ ID NO: 8 has an amino acid sequence in which the cysteine of PRT798 (SEQ ID NO: 6) is substituted with threonine.
  • An artificial polypeptide having the amino acid sequence represented by SEQ ID NO: 9 has an isoleucine deleted from the amino acid sequence consisting of amino acid residues 1 to 292 from the N-terminus of PRT798 (SEQ ID NO: 6), It has an amino acid sequence in which leucine is substituted with alanine or glycine.
  • An artificial polypeptide having the amino acid sequence represented by SEQ ID NO: 10 has an isoleucine deleted from the amino acid sequence consisting of amino acid residues 1 to 292 from the N-terminus of PRT798 (SEQ ID NO: 6), It has an amino acid sequence in which leucine or valine is substituted with alanine or glycine.
  • the artificial polypeptide (PRT856) having the amino acid sequence represented by SEQ ID NO: 11 has an amino acid sequence in which a region not including the N-terminal tag sequence and hinge sequence (SEQ ID NO: 17) of PRT835 (SEQ ID NO: 9) is repeated.
  • the artificial polypeptide (PRT854) having the amino acid sequence represented by SEQ ID NO: 12 has an amino acid sequence in which a region not including the N-terminal tag sequence and hinge sequence (SEQ ID NO: 17) of PRT836 (SEQ ID NO: 10) is repeated.
  • the artificial polypeptide (PRT840) having the amino acid sequence represented by SEQ ID NO: 13 is a substitution of 3 amino acid residues with respect to the amino acid sequence consisting of amino acid residues 1 to 246 from the N-terminus of PRT836 (SEQ ID NO: 10). And an amino acid sequence in which the amino acid sequence consisting of GAAAAAAG is inserted by amino acid substitution.
  • the artificial polypeptide (PRT855) having the amino acid sequence represented by SEQ ID NO: 14 has an amino acid sequence in which a region not including the N-terminal tag sequence and hinge sequence (SEQ ID NO: 17) of PRT840 (SEQ ID NO: 13) is repeated.
  • An artificial polypeptide (PRT837) having the amino acid sequence represented by SEQ ID NO: 15 comprises a tag sequence and a hinge sequence (SEQ ID NO: 17) in order from the N-terminus, a Nephila clavies spider silk protein (GenBank accession number: P46804.1, GI).
  • leucine was substituted with alanine or glycine for the modified amino acid sequence consisting of 142 amino acid residues and the amino acid sequence consisting of amino acid residues 293 to 427 from the N-terminal of PRT798 (SEQ ID NO: 6). It has an amino acid sequence formed by fusing amino acid sequences.
  • the artificial polypeptide (PRT838) having the amino acid sequence represented by SEQ ID NO: 16 has an amino acid sequence in which alanine in PRT837 (SEQ ID NO: 15) is substituted with valine.
  • the pET-22b (+) expression vector obtained in (1) was transformed into E. coli BLR (DE3) strain.
  • the transformed E. coli strain was cultured for 15 hours in 2 mL of LB medium containing ampicillin. Thereafter, the culture, OD 600 on the medium for the seed culture 100 mL (Table 2) containing ampicillin was added to a 0.005.
  • the culture temperature was kept at 30 ° C., and flask culture was performed until the OD 600 reached 5 (about 15 hours), thereby obtaining a seed culture solution.
  • the seed culture solution was added to a jar fermenter to which 500 mL of production medium (Table 3) was added so that the OD 600 was 0.05, and main culture was performed.
  • the culture temperature was maintained at 37 ° C.
  • the pH of the medium was controlled to be constant at 6.9
  • the dissolved oxygen concentration in the medium was maintained at 20% of the dissolved oxygen saturation concentration.
  • a feed solution (glucose 455 g / 1 L, yeast extract 120 g / 1 L) was added at a rate of 1 mL / min. Subsequently, the main culture was continued for 20 hours under the same conditions as before the addition of the feed solution.
  • IPTG 1M isopropyl- ⁇ -thiogalactopyranoside
  • the target polypeptide artificial polycrystal having the amino acid sequence shown in SEQ ID NOs: 6 to 16
  • Peptide was induced to express.
  • the culture solution was centrifuged to recover the cells.
  • SDS-PAGE was performed using bacterial cells prepared from the culture solution before and after the addition of IPTG, and the expression of the artificial polypeptide was confirmed by the appearance of a band of the desired polypeptide size depending on the addition of IPTG.
  • FIG. 2 shows the expression of PRT798 (SEQ ID NO: 6) (molecular weight 54 kDa), PRT800 (SEQ ID NO: 7) (molecular weight 54 kDa), and PRT801 (SEQ ID NO: 8) (molecular weight 54 kDa) by SDS-PAGE electrophoresis. It is a photograph which shows the result. As shown in FIG. 2, all were able to be detected at the position estimated from the theoretical molecular weight.
  • FIG. 3 shows PRT835 (SEQ ID NO: 9) (molecular weight 30 kDa), PRT838 (SEQ ID NO: 16) (molecular weight 29 kDa), PRT840 (SEQ ID NO: 13) (molecular weight 27 kDa), PRT836 (SEQ ID NO: 10) (molecular weight 29 kDa).
  • FIG. 3 is a photograph showing the results of analyzing the expression of an artificial polypeptide of PRT837 (SEQ ID NO: 15) (molecular weight 29 kDa) by SDS-PAGE electrophoresis. As shown in FIG. 3, a band was confirmed on the polymer side of the theoretical molecular weight because of the high hydrophobicity of these artificial polypeptides.
  • FIG. 4 is a photograph showing the results of SDS-PAGE electrophoresis analysis of the expression of artificial polypeptides of PRT854 (SEQ ID NO: 12), PRT855 (SEQ ID NO: 14), and PRT856 (SEQ ID NO: 11). As shown in FIG. 4, these artificial polypeptides also showed bands on the polymer side from the theoretical molecular weight.
  • the cells were treated twice at room temperature and a pressure of 600 bar to disrupt the cells. After crushing, the crushed cells were treated with a centrifuge (refrigerated centrifuge Model 7000, manufactured by Kubota Shoji Co., Ltd.) at 11,000 ⁇ g for 20 minutes to obtain an insoluble material. Furthermore, the above procedure was repeated to obtain an insoluble material.
  • a centrifuge refrigerated centrifuge Model 7000, manufactured by Kubota Shoji Co., Ltd.
  • the obtained insoluble material was washed with 20 mM Tris-HCl buffer (pH 7.4) until the purity became high.
  • the insoluble material after washing is suspended in 8 M guanidine buffer (8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) at a concentration of 100 mg / mL at 60 ° C. Stir with a stirrer for 30 minutes to dissolve. After dissolution, the suspension was dialyzed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated polypeptide obtained after dialysis was collected by centrifugation, the water was removed with a freeze dryer, and a lyophilized powder of the artificial polypeptide was collected.
  • 8 M guanidine buffer 8 M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1
  • the water content of the freeze-dried powder was measured using a hybrid Karl Fischer water analyzer (MKH-700, manufactured by Kyoto Electronics Industry Co., Ltd.).
  • the viscosity of the dope solution was measured using an electromagnetic spinning viscometer (manufactured by Kyoto Electronics Industry Co., Ltd.).
  • the viscosity of the dope solution measured in the temperature range of 40 to 50 ° C. under sealing and at a rotational speed of 1000 rpm was 1000 to 1500 cP.
  • the dope solution prepared above was filtered through a metal filter having a mesh size of 3 ⁇ m, filled in a syringe heated to 40 ° C., and degassed for 1 hour in a state where nitrogen gas was circulated and maintained at 40 ° C. After deaeration, the dope solution was discharged into a 100% methanol coagulation bath from a syringe having a nozzle hole having a diameter of 0.25 mm. At the time of discharge, the nozzle tip of the syringe was installed so as to have a gap between the surface of the coagulation solvent. The fibers obtained by coagulation were washed through two water baths and dried through a dry heat plate. The obtained artificial polypeptide fiber (keratin type fiber) was wound up with a winder. The total draw ratio was 1.7 times.
  • the mechanical strength of the artificial polypeptide fiber is determined by placing the artificial polypeptide fiber in a constant temperature and humidity chamber (LHL-113, manufactured by Espec Co., Ltd.) at 20 ° C. and a relative humidity of 65% for 24 hours. Measurement was performed using FORCE TRANSDUCER 2519-101 (registered trademark). As a comparative example, natural wool was measured under the same conditions.
  • the diameter of the filament was measured using a microscope (Nikon Corporation, ECLIPSE LV 100ND).
  • the artificial polypeptide fiber (keratin type fiber) according to the present invention has a fiber diameter almost the same as that of natural wool having a complicated structure and substantially the same mechanical characteristics.

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